Breast cancer is the most common cancer among women in industrialized countries. The development of breast cancer is a multiple-step process and regulated by the tumor microenvironment. This development process may take many years and is difficult to follow in vivo. Therefore, there is a need to develop in vitro models to study the molecular basis of tumorigenesis and progression in breast cancer as well as in other cancers.
Most in vitro cancer cell studies use standard two-dimensional (2D) cell culture systems. However, cells grown on 2D tissue culture behave differently from those grown in a physiological three-dimensional (3D) environment due to the lack of proper cell-cell and cell-matrix interactions as well as the lack of gradient of nutrients and growth factors, which are known to play critical roles in cancer initiation, progression and metastasis. For example, when cancer cells are cultured in 2D plates, their malignancy is reduced compared to those under in vivo conditions. Animal models are also frequently used to study molecular pathways and drug response in cancer research. In these cases, either animal tumors grown in syngeneic animals or human tumors grown in immunocompromised animals are used. Therefore, animal models may not adequately reproduce the features of human cancers in vivo.
To bridge the gap between the 2D cell culture system and the in vivo system, the 3D in vitro cell culture system has emerged. In many 3D models, cell lines or cells from dissociated tissues are embedded in 3D matrices and cultured to promote cell-cell interaction, adhesion, migration and in vivo-like morphogenesis. Comparison between 2D and 3D culture systems has revealed significant differences in all aspects of cell behavior from cell shape and growth to gene expression and response to stimuli. Various types of materials have been used to generate a 3D matrix. Type 1 collagen and Matrigel™ are the most widely used matrices because they are biocompatible and support adhesion and growth of many cell types. Alginate and agarose gels are also used as a matrix to study the behavior of cancer cells under 3D conditions. Unfortunately, it is difficult to control the physical characteristics of gels formed of naturally derived polymers. In addition, it is difficult to isolate and study cell response to individual factors in these microenvironments as the naturally derived matrices tend to interact with surface receptors of the cells.
As a result, inert synthetic polymers have been examined for use in development of 3D gels. The use of inert synthetic polymers can provide increased flexibility in designing 3D matrices with a wide range of mechanical, physical, and biological properties. Among the synthetic materials, polyethylene glycol (PEG) hydrogel, due to its inert nature, has been used extensively to form engineered matrices for cell encapsulation. While the development of PEG 3D matrices has been an improvement in the art, room for further improvement exists.
For example, while the effect of matrix stiffness on the response of normal stem cells has been studied, the effect of matrix stiffness on cancer stem cells (CSCs) encapsulated within an inert microenvironment has not been investigated. Normal stem cells and cancer stem cells use similar signaling pathways to maintain their stemness. However, they may respond to the environmental cues differently. The microenvironment or niche under normal conditions inhibits stem cell proliferation and differentiation, but cancer stem cells, due to mutations in the cell, are self-sufficient with respect to proliferation. It has been proposed that the stem cell niche is converted from proliferation inhibitory to one favoring cell proliferation in the case of cancer stem cells. What is needed in the art is a tunable 3D matrix that can be utilized to examine such propositions for further understanding the growth and development of cancer cells, and in particular cancer stem cells, e.g., a 3D matrix that can be utilized to enrich a cell sample in cancer stem cells. For instance, the fraction of CSCs in the population of cancer cells is understood to be at most a few percent, and possibly less than 1%. As a result, drug toxicity tests to date evaluate the response of non-stem-like cancer cells to the chemotherapy agent. Unfortunately, CSC's are the cell fraction responsible for cancer recurrence, relapse, and metastasis and the CSC fraction is the most malignant fraction of cells in the population of cancer cells. Therefore, there is a need to develop technologies and 3D matrices that can be utilized to enrich a cell population in cancer stem cells for study and drug testing.
In addition to the need to develop improved 3D matrices, as cancer cells are affected by many factors in their microenvironment, another major challenge to understanding the growth and development of cancer cells lies in developing methods to isolate the effect of single factors on particular cell types while keeping other factors unchanged. For instance, breast tumors are highly heterogeneous, and cells with self-renewal and highly invasive capacity coexist with cells that are more differentiated and non-invasive. Increasing evidence suggests that the heterogeneity of the tumor tissue is rooted in the existence of CSCs. Therefore, understanding the mechanism of CSC maintenance, and in particular the effect of specific factors on CSC maintenance and enrichment, is critical for breast cancer prevention and treatment.
Cell to cell interactions between stem cells and support cells, interactions between stem cells and extracellular matrix (ECM), the composition of ECM and the physicochemical properties of the environment are all key contributing factors in stem cell maintenance. Many in vitro studies have provided insight on the regulation of CSC fate by the microenvironment. However, these studies have been limited by the nature of the support matrix as well as by the inability to isolate the effect of single factors in a realistic model. Accordingly, what is also needed in the art is a 3D matrix having a highly controlled microenvironment so as to more accurately isolate and determine the effects of particular factors on the growth and development of cancer cells, and in particular, of stem cancer cells.